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Stage 0 sporulation gene A (spo0A) as a molecular marker to study diversity of endospore-forming
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Firmicutes
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Tina Wunderlin, Thomas Junier, Ludovic Roussel-Delif, Nicole Jeanneret, Pilar Junier*
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Laboratory of Microbiology, Institute of Biology, University of Neuchatel, CH-2000, Neuchâtel, Switzerland
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*
Corresponding author: E-mail: pilar.junier@unine.ch
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Supplementary Information
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Experimental procedures
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DNA extraction procedure
DNA from sediment samples was extracted using three different protocols:
Protocol 1) Standard extraction with in situ lysis in 0.5 g sediment using the MP FastDNA® SPIN Kit for Soil
(MP Biomedicals, Santa Ana, CA, USA), following the manufacturer’s instructions.
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Protocol 2) Repeated extraction using MP FastDNA® SPIN kit with the following modifications: 0.5 g
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sediment was subjected to three repetitive extractions with in situ lysis using bead-beating at 50 strokes per
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second with the TissueLyser LT (QIAGEN, Hilden, Germany) for 10 min. The sample was then centrifuged and
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900 µl of supernatant fluid was collected in a separate tube. Lysis buffer was again added to the samples before
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subjecting to a second round of bead-beating for 5 min, then centrifuged and supernatant fluid collected. This
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procedure was repeated a third time. The three supernatants were then processed separately following the
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standard protocol. Finally, the three extractions were pooled and DNA precipitated with 0.3 M Na-acetate and
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ethanol (99%) and washed with ethanol (70%) before being re-suspended in sterile water.
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Protocol 3) Indirect extraction, separating biomass from sediment particles prior to lysis. Three grams of
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sediment were homogenized with 15 ml of dispersing agent (1 % Na-hexa-meta-phosphate) using an Ultra-
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Turrax homogenizer (IKA, Staufen, Germany) at 15500 rpm for two minutes to separate cells from the
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sediment matrix. Coarse particles were then removed from the slurry by centrifugation at 20 x g for 1 minute,
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and the supernatant (containing the cells) was collected on a nitrocellulose membrane of 0.2 µm pore size
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(Whatman, Dassel, Germany). The cell separation step was then repeated. Filters were immediately frozen in
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liquid nitrogen and stored at -80°C. DNA was then extracted directly from the membrane with Protocol 2.
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DNA yield was measured with a Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using the Quant-iT
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dsDNA BR assay kit, following the manufacturer’s instructions. DNA quality was verified by agarose gel
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electrophoresis and by spectrophotometer absorbance at 260 and 230 nm using NanoDrop ND-1000
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(NanoDrop, Wilmington, DE, USA).
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Quantification of gene copy numbers
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Quantification of bacterial DNA in sediment extracts was performed by real-time quantitative PCR of the V3
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region of the 16S rRNA gene with primers 338f and 520r (Ovreås et al., 1997). The qPCR mix contained 1 μL of
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10-fold diluted DNA template (1.3 to 8.4 ng/µL), 0.3 μM of each primer and 10 μL of QuantiTect SYBR® Green
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PCR Kit (QIAGEN). Total reaction volume of 20 μL was reached with PCR-grade water. The qPCR was run with
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a Rotor-GeneTM 6000 instrument (QIAGEN) with the program: enzyme activation at 95°C for 5 min, 40 cycles
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of denaturation at 95°C for 5 sec, annealing at 55°C for 15 sec and extension at 72 °C for 20 sec. Thresholds
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(Th), Ct values, and derivatives of melting curves were determined using Rotor-Gene 6 software. All extracts
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were analyzed in triplicate. For quantification three independent plasmid standards series with 300 to 3 000
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000 gene copies/µL of the 16S rRNA gene of an environmental clone were included.
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Quantification of spo0A gene was performed as mentioned above for the 16S rRNA gene but with the
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primers spo0A655f and spo0A923r (Bueche et al., 2013). The qPCR mix contained 1 μL of 10-fold diluted DNA
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sample (1.3 to 8.4 ng/µL), 0.76 μM of each primer and 1 x QuantiTect SYBR® Green PCR Kit. Total reaction
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volume of 20 μL was reached with PCR-grade water. The program differed in an annealing at 52°C for 30 sec
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and extension at 72 °C for 30 sec. For quantification three independent plasmid standards series with 30 to 300
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000 gene copies/µL of spo0A gene of B. subtilis were included.
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Amplification and sequencing of the spo0A gene
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One extract from each DNA extraction protocol from both sediments (Lake Geneva and Baikal) was
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subjected to amplicon sequencing of the spo0A gene.
Degenerate primers amplifying a 602 bp sequence of the spo0A gene were designed for this study. Primer
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sequences were spo0A166f (5’-GATATHATYATGCCDCATYT-3’) and spo0A748r (5’-
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GCNACCATHGCRATRAAYTC-3’). PCR reactions were performed with 0.5 ng DNA template, 1 x reaction buffer
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(TaKaRa Bio, Shiga, Japan), 3 mM MgCl2, 10 µg bovine serum albumin (BSA; New England Biolabs, Ipswich, MA,
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USA), 1 U of Ex Taq Polymerase (TaKaRa), 200 µM of each dNTP and 1 µM of each primer in a total reaction
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volume of 50 µl, completed with PCR-grade water. Negative controls (1 µl PCR-grade water) and positive
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controls (1 ng Paenibacillus alvei DNA template) were included in all reactions. Reactions were run on an
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Arktik Thermo Cycler (Thermo Fisher Scientific, Vantaa, Finland) with the following temperature program:
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initial denaturation at 94°C for 5 min; then 10 cycles of denaturation at 94°C for 30 min, touchdown annealing
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starting at 55°C with decrease of 0.3°C per cycle for 30s and elongation at 72°C for 1 min; followed by 30 cycles
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of denaturation at 94°C for 30 min, annealing at 52°C for 30s and elongation at 72°C for 1 min; and a final
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extension at 72°C for 5 min. Prior to the amplification of environmental samples, the primers were tested on
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pure strains of endospore-forming and non sporulating bacteria (Supplementary Table 2).
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Amplified sediment samples were sent for barcode amplicon sequencing with Roche GS FLX+ (Eurofins
MWG Operon, Ebersberg, Germany).
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Supplementary Figure 1. Comparative phylogenetic analysis of 16S rRNA gene sequences and six conserved
sporulation-related genes (spo0A, spoIVB, spoVAC, spoVAD, spoVT and gpr)(spore proteome) for 27 sporeforming Firmicutes with a complete genome sequence reported and annotated. Alignments were constructed
with MAFFT (Katoh et al. 2005) or Muscle (Edgar 2004) using default parameters. Multiple-FastA alignments
were converted to Phylip format with the seqret program from the EMBOSS package (Rice et al. 2000).
Phylogenies were constructed from Phylip-formatted alignments with PhyML (Guindon and Gascuel 2003),
using default parameters, except the following: JTT+Γ substitution model for proteins and GTR+ Γ model for
nucleic acids; 4 classes of substitution rate categories; estimation of the shape parameter, proportion of
invariants, and transition/transversion ratios (for nucleotides). Trees were processed (re-rooting, extracting
topology, and plotting) with the Newick Utilities (Junier and Zdobnov 2010). Bootstrap values (percentage over
1000 samplings) are shown at the nodes of the trees.
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Supplementary Figure 2. Phylogenetic reconstruction (above) and conservation profiles (below) for
sequences of the stage 0 sporulation protein Spo0A. Conservation plots were made with the plotcon program
from EMBOSS. This is a sliding-window program that computes a weighted average of the similarity scores for
all residue pairs in each window. We used the default window size of 4 residues.
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Supplementary Figure 3. Alignment of spo0A gene of Sulfobacillus acidophilus and Alicyclobacillus
acidocaldarius Tc41 against spo0A of Bacillus subtilis 168. The two regions shown correspond to the forward
primer 166f (left) and the reverse primer 748r (right) described in this study. Stars indicate 100% identity.
The exclamation points highlight mismatches with the primer sequence.
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Supplementary Figure 4. Cladogram of spo0A sequences from sediment of Lake Geneva extracted with
protocols 1 (blue), 2 (yellow), and 3 (red).. The nucleotide sequences were then clustered into putative OTUs
(identity of > 97%) with the pick_otus.py program from the QIIME package using the Uclust method
(Caporaso, 2010 #87), and a representative was used to build the phylogeny. Phylogenies were constructed
from Phylip-formatted alignments with PhyML (Guindon, 2003 #88), using default parameters. The trees were
re-rooted, condensed according to DNA extraction protocol, and displayed with the Newick utilities (Junier,
2010 #4). Each branch represents a cluster of OTUs of > 97% sequence similarity. Identification of the closest
relatives of the environmental sequences from the indirect extractions (protocol 3) was done by protein BLAST
(Altschul et al., 1997) with the translated protein sequences using a reference database of 581 Spo0A protein
sequences from the InterPro site (Mulder et al. 2002). Classes of closest relative are shown in color with
indication of the identity ranges (<65% identity (-), 65-74% (<), 75-84% (~), 85-94%(#), >95% (+)). A Bacillus
amyloliquefaciens, B B. methanolicus, C Geobacillus sp. (strain WCH70), D B. cereus subsp. cytotoxis (strain NVH
391-98), E B. thuringiensis , F Geobacillus thermodenitrificans (strain NG80-2), G B. atrophaeus (strain 1942), H
B. subtilis, I B. mycoides, J B. pseudofirmus (strain OF4), K Lysinibacillus sphaericus (strain C3-41), L Brevibacillus
laterosporus, M Brevibacillus brevis (strain 47), N Thermincola potens (strain JR), O Desulfotomaculum
acetoxidans (strain ATCC 49208), P Desulfosporosinus orientis (strain ATCC 19365), Q Thermosediminbacter
oceani (strain ATCC BAA-1034), R Syntrophobotulus glycolicus (strain DSM 8271), S Heliobacterium
medesticaldum (strain ATCC 51547), T Clostridium clariflavum (strain DSM 19732), U B. cereus , V C.
thermocellum, W C. cellulovorans (strain ATCC 35296), X C. cellulolyticum (strain ATCC 35319), Y C. botulinum ,
Z C. lijungdahlii (strain ATCC 55383), AA C. perfringens, AB C. sporogenes , AC Alkaliphilus metalliredigens
(strain QYMF), AD A. oremlandii (strain OhILAs), AE Desulfotomaculum kuznetsovii (strain DSM 6115), AF
Geobacillus sp. (strain Y412MC10), AG Paenibacillus polymyxa , AH P. mucilaginosus (strain KNP414).
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Supplementary Figure 5. Cladogram of spo0A sequences from sediment of Lake Baikal extracted with
protocols 1 (blue), 2 (yellow), and 3 (red). Each branch represents a cluster of OTUs of > 97% sequence
similarity. Closest relatives are shown in letters around the tree together with identity ranges (<65% identity (), 65-74% (<), 75-84% (~), 85-94%(#), >95% (+)). For classes see legend in Figure 4 and the following: AI B.
megaterium, AJ B. licheniformis , AK B. megaterium (strain DSM 319) AL C. haemolyticum, AM Paenibacillus sp.
(strain JDR-2), AN B. cellulosilyticus (strain ATCC 21833), AO Sulfobacillus acidophilus (strain TPY), AP S.
acidophilus (strain ATCC 700253), AQ Desulforudis audaxviator (strain MP104C), AR C. butyricum , AS C.
kluyveri (strain ATCC 8527).
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Supplementary Table 1. List of genome sequences from the 27 endospore-forming Firmicutes used in this study. Complete and draft genome sequences were
downloaded from the Comprehensive Microbial Resource (CMR, 24.0 data release, cmr.jcvi.org) and Integrated Microbial Genomes (IMG, 3.0, img.jgi.doe.gov)
websites. Protein and nucleotide sequences of spore-related genes were obtained by search for role category/function sporulation and germination (CMR) and
sporulating (IMG). Additional information on all retrieved genomes was obtained from the GenBank database (www.ncbi.nlm.nih.gov/genome). Clas= taxonomical
classification; B= Bacilli; C= Clostridia; T°= temperature range; M= mesophile; T= thermophile; P= psychrophile; H= hyperthermophile; Sp. Genes= number of
sporulation-related genes. The number of sporulation-related genes was retrieved from the available genome annotations.
Name
Bacillus amyloliquefaciens FZB42
Bacillus anthracis A0248
Bacillus anthracis Sterne
Bacillus cereus 03BB102
Bacillus cereus Zk
Bacillus licheniformis ATCC 14580 (Novozymes)
Bacillus pumilus SAFR-032
Bacillus subtilis 168
Bacillus thuringiensis Al Hakam
Bacillus weihenstephanensis KBAB4
Geobacillus thermodenitrificans NG80-2
Lysinibacillus sphaericus C3-41
Alkaliphilus metalliredigens QYMF
Alkaliphilus oremlandii OhILAs
Candidatus Desulforudis audaxviator MP104C
Carboxydothermus hydrogenoformans Z-2901
Clostridium beijerinckii NCIMB 8052
Clostridium botulinum A2 Kyoto-F
Clostridium botulinum B Eklund 17B
Clostridium difficile 630
Clostridium kluyveri DSM 555
Clostridium perfringens SM101
Heliobacterium modesticaldum Ice1
Pelotomaculum thermopropionicum SI
Thermoanaerobacter pseudethanolicus ATCC 33223
Thermoanaerobacter sp. X514
Desulfotomaculum reducens MI-1
Taxon ID
326423
592021
260799
572264
288681
279010
315750
224308
412694
315730
420246
444177
293826
350688
477974
246194
290402
536232
508765
272563
431943
289380
498761
370438
340099
399726
349161
Clas.
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Isolation
Soil
Human isolate
Soil
Human blood
Wab of a dead zebra carcass
Soil
Soil
X-ray irradiated strain
Severe human tissue necrosis
Soil
Water in oil reservoir formation
Mosquito breeding site
Borax leachate ponds
Freshwater USA
Fracture water from a borehole
Hot swamp
Freshwater, Soil
Infant botulism
Marine sediments
Clinical isolate
Mud of a canal in Delft
Soil
Hot spring microbial mats and volcanic soil
Thermophilic anaerobic sludge
Thermal springs
Deep subsurface sample
Cr-contaminated marine sediment
T°
M
M
M
M
M
M
M
M
M
P
T
M
M
M
M
H
M
M
M
M
M
M
T
T
T
T
M
Genes
3814
5418
5521
5767
5134
4420
3823
4298
5050
5983
3642
4887
5016
2951
2293
2707
5290
3978
3639
3983
4073
2748
3142
3018
2363
2467
3324
CDS
3696
5291
5287
5621
5323
4196
3729
4106
4798
5831
3471
4771
4801
2836
2239
2645
5100
3878
3527
3777
3913
2578
3001
2920
2291
2397
3324
rRNA
30
33
33
42
39
21
21
30
42
42
30
31
31
26
6
12
43
20
34
32
20
30
30
6
16
16
18
GC Perc
0.46
0.35
0.35
0.35
0.35
0.46
0.41
0.44
0.35
0.35
0.49
0.37
0.37
0.36
0.61
0.42
0.3
0.28
0.27
0.29
0.32
0.28
0.57
0.53
0.35
0.35
0.42
Sp. genes
111
190
82
183
75
69
117
129
102
115
101
96
83
75
63
61
62
98
82
63
77
67
82
66
76
79
83
Reference
(Chen et al. 2007)
Unpublished
Unpublished
Unpublished
(Han et al. 2006)
(Rey et al. 2004)
(Gioia et al. 2007)
(Kunst et al. 1997)
(Challacombe et al. 2007)
Unpublished
(Feng et al. 2007)
(Hu et al. 2008)
Unpublished
Unpublished
(Chivian et al. 2008)
(Wu et al. 2005)
Unpublished
Unpublished
Unpublished
(Sebaihia et al. 2006)
(Seedorf et al. 2008)
(Shimizu et al. 2002)
(Sattley et al. 2008)
Unpublished
Unpublished
Unpublished
(Junier et al. 2010)
Supplementary Table 2. Orthologous genes found after bi-directional BLAST of the sporulationrelated genes common to 27 genomes of endospore-forming Firmicutes. Protein lengths
indicated for Bacillus subtilis as a reference were obtained from Stragier & Losick (1996).
Name
Stage 0 sporulation
protein A
Spore protease
Stage V sporulation
protein T
Stage IV sporulation
protein B
Stage V sporulation
protein AD
Stage V sporulation
protein AC
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Gene
symbol
spo0A
gpr
spoVT
spoIVB
spoVAD
spoVAC
Function
Length in Bacillus
subtilis (aa)
Global transcription regulator for sporulation
267
Degradation of the small acid-soluble spore proteins
(SASPs) during germination
368
Global regulator activated by sigma G
178
Protease that activates processing of the pro-sigma
K factor
Potential transmembrane protein with unknown
function
Potential transmembrane protein with unknown
function
425
150
338
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